The archaeal plasma membrane consists mainly of diether lipids and tetraether lipids instead of the usual ester lipids found in other organisms. Although a molecule of tetraether lipid is thought to be synthesized from two molecules of diether lipids, there is no direct information about the biosynthetic pathway(s) or intermediates of tetraether lipid biosynthesis. In this study, we examined the effects of the fungal squalene epoxidase inhibitor terbinafine on the growth and ether lipid biosyntheses in the thermoacidophilic archaeon Thermoplasma acidophilum. Terbinafine was found to inhibit the growth of T. acidophilum in a concentration-dependent manner. When growing T. acidophilum cells were pulse-labeled with [2-14C]mevalonic acid in the presence of terbinafine, incorporation of radioactivity into the tetraether lipid fraction was strongly suppressed, while accumulation of radioactivity was noted at the position corresponding to diether lipids, depending on the concentration of terbinafine. After the cells were washed with fresh medium and incubated further without the radiolabeled substrate and the inhibitor, the accumulated radioactivity in the diether lipid fraction decreased quickly while that in the tetraether lipids increased simultaneously, without significant changes in the total radioactivity of ether lipids. These results strongly suggest that terbinafine inhibits the biosynthesis of tetraether lipids from a diether-type precursor lipid(s). The terbinafine treatment will be a tool for dissecting tetraether lipid biosynthesis in T. acidophilum.
The lipid composition of Candidatus “Aciduliprofundum boonei”, the only cultivated representative of archaea falling in the DHVE2 phylogenetic cluster, a group of microorganisms ubiquitously occurring at hydrothermal vents, was studied. The predominant core membrane lipids in this thermophilic euryarchaeote were found to be composed of glycerol dibiphytanyl glycerol tetraethers (GDGTs) containing 0–4 cyclopentyl moieties. In addition, GDGTs with an additional covalent bond between the isoprenoid hydrocarbon chains, so-called H-shaped GDGTs, were present. The latter core lipids have been rarely reported previously. Intact polar lipid analysis revealed that they predominantly consist of GDGTs with a phospho-glycerol headgroup.
Aciduliprofundum boonei; DHVE2 cluster; Glycerol dialkyl glycerol tetraethers; Thermoacidophile
Methanospirillum hungatei GP1 contained 50% of its ether core lipids (polar lipids less head groups) as tetraether lipids, and its plasma membrane failed to fracture along its hydrophobic domain during freeze-etching. The membrane of Methanosaeta ("Methanothrix") concilii did not contain tetraether lipids and easily fractured to reveal typical intramembranous particles. Methanococcus jannaschii grown at 50 degrees C contained 20% tetraether core lipids, which increased to 45% when cells were grown at 70 degrees C. The frequency of membrane fracture was reduced as the membrane-spanning tetraether lipids approached 45%. As the tetraether lipid content increased, and while fracture was still possible, the particle density in the membrane increased; these added particles could be tetraether lipid complexes torn from the opposing membrane face. The diether membrane (no tetraether lipid) of Methanococcus voltae easily fractured, and the intramembranous particle density was low. Protein-free liposomes containing tetraether core lipids (ca. 45%) also did not fracture, whereas those made up exclusively of diether lipids did split, indicating that tetraether lipids add considerable vertical stability to the membrane. At tetraether lipid concentrations below 45%, liposome bilayers fractured to reveal small intramembranous particles which we interpret to be tetraether lipid complexes.
Glycerol dialkyl glycerol tetraethers (GDGTs) are core membrane lipids originally thought to be produced mainly by (hyper)thermophilic archaea. Environmental screening of low-temperature environments showed, however, the abundant presence of structurally diverse GDGTs from both bacterial and archaeal sources. In this study, we examined the occurrences and distribution of GDGTs in hot spring environments in Yellowstone National Park with high temperatures (47 to 83°C) and mostly neutral to alkaline pHs. GDGTs with 0 to 4 cyclopentane moieties were dominant in all samples and are likely derived from both (hyper)thermophilic Crenarchaeota and Euryarchaeota. GDGTs with 4 to 8 cyclopentane moieties, likely derived from the crenarchaeotal order Sulfolobales and the euryarchaeotal order Thermoplasmatales, are usually present in much lower abundance, consistent with the relatively high pH values of the hot springs. The relative abundances of cyclopentane-containing GDGTs did not correlate with in situ temperature and pH, suggesting that other environmental and possibly genetic factors play a role as well. Crenarchaeol, a biomarker thought to be specific for nonthermophilic group I Crenarchaeota, was also found in most hot springs, though in relatively low concentrations, i.e., <5% of total GDGTs. Its abundance did not correlate with temperature, as has been reported previously. Instead, the cooccurrence of relatively abundant nonisoprenoid GDGTs thought to be derived from soil bacteria suggests a predominantly allochthonous source for crenarchaeol in these hot spring environments. Finally, the distribution of bacterial branched GDGTs suggests that they may be derived from the geothermally heated soils surrounding the hot springs.
The present studies were focused on the formation and characterization of sterically stabilized archaeosomes made from a synthetic PEGylated archaeal lipid. In a first step, a synthetic archaeal tetraether bipolar lipid was functionalized with a poly(ethylene glycol), PEG, and (PEG45-Tetraether) with the aim of coating the archaeosome surface with a sterically stabilizing hydrophilic polymer. In a second step, Egg-PC/PEG45-Tetraether (90/10 wt%) archaeosomes were prepared, and their physicochemical characteristics were determined by dynamic light scattering (size, polydispersity), cryo-TEM (morphology), and by high-performance thin layer chromatography (lipid composition), in comparison with standard Egg-PC/PEG45-DSPE formulations. Further, a fluorescent dye, the carboxyfluorescein, was encapsulated into the prepared archaeosomes in order to evaluate the potential of such nanostructures as drug carriers. Release studies have shown that the stability of Egg-PC/PEG45-Tetraether-based archaeosomes is significantly higher at 37°C than the one of Egg-PC/PEG45-DSPE-based liposomes, as evidenced by the slower release of the dye encapsulated into PEGylated archaeosomes. This enhanced stability could be related to the membrane spanning properties of the archaeal bipolar lipid as already described with natural or synthetic tetraether lipids.
Three bipolar archaeal-type diglycerophosphocholine tetraether lipids (a.k.a., bolalipids) have been prepared to determine 1) the influence of molecular structure on the physical properties of bolalipid membranes and 2) their impact on the functional reconstitution of Ste14p, a membrane-associated isoprenylcysteine carboxyl methyltransferase from Saccharomyces cerevisiae. The three bolalipids synthesized were: C20BAS, C32BAS, and C32phytBAS. These bolalipid structures differ in that the C20BAS derivative has a short sn-1 glyceryl diether C20H40 transmembrane alkyl chain and two ether-linked sn-2 n-decyl chains, whereas the C32BAS and C32phytBAS derivatives have a longer sn-1 diether C32H64 membrane-spanning chain and two ether-linked sn-2 n-hexadecyl or phytanyl chains, respectively. Differential scanning calorimetry and temperature-dependent 31P NMR was used to determine the gel-to-liquid crystalline phase transition temperatures of the bolalipids (C32BAS Tm > 85 °C; C32phytBAS Tm = 14 °C; C20BAS Tm = 17°C). The bolalipid lateral diffusion coefficients, determined by fluorescence recovery after photobleaching at 20 °C, were 1.5 × 10−8 and 1.8 × 10−9 cm2/s for C20BAS and C32phytBAS, respectively. The mobility of C32BAS could not be measured at this temperature. Ste14p activity was monitored by an in vitro methyltransferase assay in reconstituted vesicle dispersions composed of DMPC, C20BAS:E. coli polar lipid, C20BAS:POPC, C32phytBAS:E. coli polar lipid, and C32phytBAS:POPC. Ste14p activity was lost in vesicles composed of 75–100 mol% C20BAS and 0–100 mol% C32BAS, but retained in vesicles with 0–50 mol% C20BAS and 0–100 mol% C32phytBAS. Confocal immunofluorescence microscopy confirmed the presence of Ste14p in 100 mol% C20BAS and 100 mol% C32phytBAS vesicle dispersions, even though the lamellar liquid crystalline phase thickness of C20BAS is only 32 Å. Since Ste14p activity was not affected by either the gel to liquid-crystal phase transition temperature of the lipid or the temperature of the assay, the low activity observed in 75–100 mol% C20BAS membranes can be attributed to hydrophobic mismatch between this bolalipid and the hydrophobic surface of Ste14p.
Crenarchaeol, a membrane-spanning glycerol dialkyl glycerol tetraether (GDGT) containing a cyclohexane moiety in addition to four cyclopentane moieties, was originally hypothesized to be synthesized exclusively by the mesophilic Crenarchaeota. Recent studies reporting the occurrence of crenarchaeol in hot springs and as a membrane constituent of the recently isolated thermophilic crenarchaeote “Candidatus Nitrosocaldus yellowstonii,” however, have raised questions regarding its taxonomic distribution and function. To determine whether crenarchaeol in hot springs is indeed synthesized by members of the Archaea in situ or is of allochthonous origin, we quantified crenarchaeol present in the form of both intact polar lipids (IPLs) and core lipids in sediments of two California hot springs and in nearby soils. IPL-derived crenarchaeol (IPL-crenarchaeol) was found in both hot springs and soils, suggesting in situ production of this GDGT over a wide temperature range (12°C to 89°C). Quantification of archaeal amoA gene abundance by quantitative PCR showed a good correspondence with IPL-crenarchaeol, suggesting that it was indeed derived from living cells and that crenarchaeol-synthesizing members of the Archaea in our samples may also be ammonia oxidizers.
Glycerol dibiphytanyl glycerol tetraether (GDGT)-based intact membrane lipids are increasingly being used as complements to conventional molecular methods in ecological studies of ammonia-oxidizing archaea (AOA) in the marine environment. However, the few studies that have been done on the detailed lipid structures synthesized by AOA in (enrichment) culture are based on species enriched from nonmarine environments, i.e., a hot spring, an aquarium filter, and a sponge. Here we have analyzed core and intact polar lipid (IPL)-GDGTs synthesized by three newly available AOA enriched directly from marine sediments taken from the San Francisco Bay estuary (“Candidatus Nitrosoarchaeum limnia”), and coastal marine sediments from Svalbard, Norway, and South Korea. Like previously screened AOA, the sedimentary AOA all synthesize crenarchaeol (a GDGT containing a cyclohexane moiety and four cyclopentane moieties) as a major core GDGT, thereby supporting the hypothesis that crenarchaeol is a biomarker lipid for AOA. The IPL headgroups synthesized by sedimentary AOA comprised mainly monohexose, dihexose, phosphohexose, and hexose-phosphohexose moieties. The hexose-phosphohexose headgroup bound to crenarchaeol was common to all enrichments and, in fact, the only IPL common to every AOA enrichment analyzed to date. This apparent specificity, in combination with its inferred lability, suggests that it may be the most suitable biomarker lipid to trace living AOA. GDGTs bound to headgroups with a mass of 180 Da of unknown structure appear to be specific to the marine group I.1a AOA: they were synthesized by all three sedimentary AOA and “Candidatus Nitrosopumilus maritimus”; however, they were absent in the group I.1b AOA “Candidatus Nitrososphaera gargensis.”
Branched glycerol dibiphytanyl glycerol tetraethers (bGDGTs) are known as bacterial lipids that occur widely in terrestrial environments, particularly in anaerobic peat bogs and soil. We examined the abundance and distribution of bGDGTs in both core (C) and polar (P) lipid fractions from the water column and surface sediments in the lower Pearl River (PR) and its estuary using two extraction methods (sonication vs. Bligh and Dyer). A number of soil samples in the lower PR drainage basin were also collected and extracted for bGDGTs using the sonication method. The results showed aquatic production of bGDGTs as supported by substantial abundances of P-bGDGTs in the water column and sediment samples. The bGDGT-based proxies (BIT, CBT, and MBT) were not affected by the method of extraction when C-bGDGTs were analyzed; in such case, the pHCBT of the sediments reflected the soil pH of the lower PR drainage basin, and the temperature close to the annual mean air temperature (MAT) in the lower PR basin. On the other hand, the P-bGDGT-derived proxies were inconsistent between the two methods. The P-bGDGTs (particularly those extracted using the sonication method) may not be reliable indicators of annual MATs.
Pearl River; estuary; bGDGTs; paleoclimate proxies
Glycerol dialkyl glycerol tetraethers (GDGTs) are core membrane lipids of the Crenarchaeota. The structurally unusual GDGT crenarchaeol has been proposed as a taxonomically specific biomarker for the marine planktonic group I archaea. It is found ubiquitously in the marine water column and in sediments. In this work, samples of microbial community biomass were obtained from several alkaline and neutral-pH hot springs in Nevada, United States. Lipid extracts of these samples were analyzed by high-performance liquid chromatography-mass spectrometry and by gas chromatography-mass spectrometry. Each sample contained GDGTs, and among these compounds was crenarchaeol. The distribution of archaeal lipids in Nevada hot springs did not appear to correlate with temperature, as has been observed in the marine environment. Instead, a significant correlation with the concentration of bicarbonate was observed. Archaeal DNA was analyzed by denaturing gradient gel electrophoresis. All samples contained 16S rRNA gene sequences which were more strongly related to thermophilic crenarchaeota than to Cenarchaeum symbiosum, a marine nonthermophilic crenarchaeon. The occurrence of crenarchaeol in environments containing sequences affiliated with thermophilic crenarchaeota suggests a wide phenotypic distribution of this compound. The results also indicate that crenarchaeol can no longer be considered an exclusive biomarker for marine species.
The membrane lipids of archaea are characterized by unique isoprenoid biochemistry, which typically is based on two core lipid structures, sn-2,3-diphytanylglycerol diether (archaeol) and sn-2,3-dibiphytanyldiglycerol tetraether (caldarchaeol). The biosynthetic pathway for the tetraether lipid entails unprecedented head-to-head coupling of isoprenoid intermediates by an unknown mechanism involving unidentified enzymes. To investigate the isoprenoid ether lipid biosynthesis pathway of the hyperthermophilic archaeon, Archaeoglobus fulgidus, its lipid synthesis machinery was reconstructed in an engineered E. coli strain in an effort to demonstrate, for the first time, efficient isoprenoid ether lipid biosynthesis for the production of the intermediate, digeranylgeranylglyceryl phosphate (DGGGP). The biosynthesis of DGGGP was verified using a LC/MS/MS technique and was accomplished by cloning and expressing the native E. coli gene for IPP isomerase (idi), along with the A. fulgidus genes for G1P dehydrogenase (egsA) and GGPP synthase (gps), under the control of the lac promoter. The A. fulgidus genes for GGGP synthase (GGGPS) and DGGGP synthase (DGGGPS), under the control of the araBAD promoter, were then introduced and expressed to enable DGGGP biosynthesis in vivo. This investigation established roles for four A. fulgidus genes in the isoprenoid ether lipid pathway for DGGGP biosynthesis and provides a platform useful for identification of subsequent, currently unknown, steps in tetraether lipid biosynthesis proceeding from DGGGP, which is the presumed substrate for the head-to-head coupling reaction yielding unsaturated caldarchaeol.
Archaeoglobus fulgidus; isoprenoid; ether lipid; DGGGP
There is great interest in the membrane lipids of archaea (glycerol dialkyl glycerol tetraethers [GDGTs]) as tracers of archaeal biomass because of their utility as paleoproxies and because of the biogeochemical importance of archaea. While core GDGTs (formed by hydrolysis of polar head groups of intact GDGTs after cell death) are appropriate for paleostudies, they have also been used to trace archaeal populations. Also, despite the small size (0.2 by 0.7 μm) of cultivated marine archaea, 0.7-μm glass-fiber filters (GFFs) are typically used to collect GDGTs from natural waters. We quantified both core and intact GDGTs in free-living (0.2- to 0.7-μm), suspended (0.7- to 60-μm), and aggregate (>60-μm) particle size fractions in Puget Sound (Washington State). On average, the free-living fraction contained 36% of total GDGTs, 90% of which were intact. The intermediate-size fraction contained 62% of GDGTs, and 29% of these were intact. The aggregate fraction contained 2% of the total GDGT pool, and 29% of these were intact. Our results demonstrate that intact GDGTs are largely in the free-living fraction. Because only intact GDGTs are present in living cells, protocols that target this size fraction and analyze the intact GDGT pool are necessary to track living populations in marine waters. Core GDGT enrichment in larger-size fractions indicates that archaeal biomass may quickly become attached or entrained in particles once the archaea are dead or dying. While the concentrations of the two pools were generally not correlated, the similar sizes of the core and intact GDGT pools suggest that core GDGTs are removed from the water column on timescales similar to those of cell replication, on timescales of days to weeks.
The S-layer of Caulobacter is a two-dimensional paracrystalline array on the cell surface composed of a single protein, RsaA. We have established conditions for preparation of stable, soluble protein and then efficient in vitro recrystallization of the purified protein. Efficient recrystallization and long range order could not be obtained with pure protein only, though it was apparent that calcium was required for crystallization. Recrystallization was obtained when lipid vesicles were provided, but only when the vesicles contained the specific species of Caulobacter smooth lipopolysaccharide (SLPS) that previous studies implicated as a requirement for attaching the S-layer to the cell surface. The specific type of phospholipids did not appear critical; phospholipids rather different from those present in Caulobacter membranes or archaebacterial tetraether lipids worked equally well. The source of LPS was critical; rough and smooth variants of Salmonella typhimurium LPS as well as the rough form of Caulobacter LPS were ineffective. The requirement for calcium ions for recrystallization was further evaluated; strontium ions could substitute for calcium, and to a lesser extent, cobalt, barium, manganese and magnesium ions also stimulated crystallization. On the other hand, nickel and cadmium provided only weak crystallization stimulation, and zinc, copper, iron, aluminum ions, and the monovalent potassium, sodium, and lithium ions were ineffective. The recrystallization could also be reproduced with Langmuir-Blodgett lipid monolayers at an air-water interface. As with the vesicle experiments, this was only successful when SLPS was incorporated into the lipid mix. The best method for RsaA preparation, leading to apparently monomeric protein that was stable for many months, was an extraction with a low pH aqueous solution. We also achieved recrystallization, albeit at lower efficiency, using RsaA protein solubilized by 8 M urea, a method which allows retrieval of protein from inclusions, when expressed as heterologous protein in Escherichia coli or when retrieved as shed, precipitated protein from certain mutant caulobacters. In summary, the clarification of recrystallization methods has confirmed the requirement of SLPS as a surface attachment component and suggests that its presence in a membrane-like structure greatly stimulates the extent and quality of S-layer formation. The in vitro approach allowed the demonstration that specific ions are capable of participating in crystallization and now provides an assay for the crystallization potential of modified S-layer proteins, whether they were produced in or can be secreted by caulobacters.
A methane-derived carbonate crust was collected from the recently
discovered NIOZ mud volcano in the Sorokin Trough, NE Black Sea during
the 11th Training-through-Research cruise of the R/V Professor
Logachev. Among several specific bacterial and archaeal membrane
lipids present in this crust, two novel macrocyclic diphytanyl
glycerol diethers, containing one or two cyclopentane rings, were
detected. Their structures were tentatively identified based on the
interpretation of mass spectra, comparison with previously reported
mass spectral data, and a hydrogenation experiment. This macrocyclic
type of archaeal core membrane diether lipid has so far been
identified only in the deep-sea hydrothermal vent methanogen
Methanococcus jannaschii. Here, we provide the first
evidence that these macrocyclic diethers can also contain internal
cyclopentane rings. The molecular structure of the novel diethers
resembles that of dibiphytanyl tetraethers in which biphytane chains,
containing one and two pentacyclic rings, also occur. Such tetraethers
were abundant in the crust. Compound-specific isotope measurements
revealed δ13C values of –104 to
–111‰ for these new archaeal lipids, indicating that they
are derived from methanotrophic archaea acting within anaerobic
methane-oxidizing consortia, which subsequently induce authigenic
anaerobic oxidation of methane; archaeal membrane lipids; fluid venting; microbial processes
Despite intense study over many years, the mechanisms by which water and small nonelectrolytes cross lipid bilayers remain unclear. While prior studies of permeability through membranes have focused on solute characteristics, such as size, polarity, and partition coefficient in hydrophobic solvent, we focus here on water permeability in seven single component bilayers composed of different lipids, five with phosphatidylcholine headgroups and different chain lengths and unsaturation, one with a phosphatidylserine headgroup, and one with a phosphatidylethanolamine headgroup. We find that water permeability correlates most strongly with the area/lipid and is poorly correlated with bilayer thickness and other previously determined structural and mechanical properties of these single component bilayers. These results suggest a new model for permeability that is developed in the accompanying theoretical paper in which the area occupied by the lipid is the major determinant and the hydrocarbon thickness is a secondary determinant. Cholesterol was also incorporated into DOPC bilayers and X-ray diffuse scattering was used to determine quantitative structure with the result that the area occupied by DOPC in the membrane decreases while bilayer thickness increases in a correlated way because lipid volume does not change. The water permeability decreases with added cholesterol and it correlates in a different way from pure lipids with area per lipid, bilayer thickness, and also with area compressibility.
The distribution of membrane lipids of 17 different strains representing 13 species of subdivisions 1 and 3 of the phylum Acidobacteria, a highly diverse phylum of the Bacteria, were examined by hydrolysis and gas chromatography-mass spectrometry (MS) and by high-performance liquid chromatography-MS of intact polar lipids. Upon both acid and base hydrolyses of total cell material, the uncommon membrane-spanning lipid 13,16-dimethyl octacosanedioic acid (iso-diabolic acid) was released in substantial amounts (22 to 43% of the total fatty acids) from all of the acidobacteria studied. This lipid has previously been encountered only in thermophilic Thermoanaerobacter species but bears a structural resemblance to the alkyl chains of bacterial glycerol dialkyl glycerol tetraethers (GDGTs) that occur ubiquitously in peat and soil and are suspected to be produced by acidobacteria. As reported previously, most species also contained iso-C15 and C16:1ω7C as major fatty acids but the presence of iso-diabolic acid was unnoticed in previous studies, most probably because the complex lipid that contained this moiety was not extractable from the cells; it could only be released by hydrolysis. Direct analysis of intact polar lipids in the Bligh-Dyer extract of three acidobacterial strains, indeed, did not reveal any membrane-spanning lipids containing iso-diabolic acid. In 3 of the 17 strains, ether-bound iso-diabolic acid was detected after hydrolysis of the cells, including one branched GDGT containing iso-diabolic acid-derived alkyl chains. Since the GDGT distribution in soils is much more complex, branched GDGTs in soil likely also originate from other (acido)bacteria capable of biosynthesizing these components.
In this study we analyzed the membrane lipid composition of “Candidatus Nitrosopumilus maritimus,” the only cultivated representative of the cosmopolitan group I crenarchaeota and the only mesophilic isolate of the phylum Crenarchaeota. The core lipids of “Ca. Nitrosopumilus maritimus” consisted of glycerol dialkyl glycerol tetraethers (GDGTs) with zero to four cyclopentyl moieties. Crenarchaeol, a unique GDGT containing a cyclohexyl moiety in addition to four cyclopentyl moieties, was the most abundant GDGT. This confirms unambiguously that crenarchaeol is synthesized by species belonging to the group I.1a crenarchaeota. Intact polar lipid analysis revealed that the GDGTs have hexose, dihexose, and/or phosphohexose head groups. Similar polar lipids were previously found in deeply buried sediments from the Peru margin, suggesting that they were in part synthesized by group I crenarchaeota.
Distributions and isotopic analyses of lipids from sediment cores at a hydrothermally active site in the Guaymas Basin with a steep sedimentary temperature gradient revealed the presence of archaea that oxidize methane anaerobically. The presence of strongly 13C-depleted lipids at greater depths in the sediments suggests that microbes involved in anaerobic oxidation of methane are present and presumably active at environmental temperatures of >30°C, indicating that this process can occur not only at cold seeps but also at hydrothermal sites. The distribution of the membrane tetraether lipids of the methanotrophic archaea shows that these organisms have adapted their membrane composition to these high environmental temperatures.
Intact core tetraether membrane lipids of marine planktonic Crenarchaeota were quantified in water column-suspended particulate matter obtained from four depth intervals (∼70, 500, 1,000 and 1,500 m) at seven stations in the northwestern Arabian Sea to investigate the distribution of the organisms at various depths. Maximum concentrations generally occurred at 500 m, near the top of the oxygen minimum zone, and the concentrations at this depth were, in most cases, slightly higher than those in surface waters. In contrast, lipids derived from eukaryotes (cholesterol) and from eukaryotes and bacteria (fatty acids) were at their highest concentrations in surface waters. This indicates that these crenarchaeotes are not restricted to the photic zone of the ocean, which is consistent with the results of recent molecular biological studies. Since the Arabian Sea has a strong oxygen minimum zone between 100 and 1,000 m, with minimum oxygen levels of <1 μM, the abundance of crenarchaeotal membrane lipids at 500 m suggests that planktonic Crenarchaeota are probably facultative anaerobes. The cell numbers we calculated from the concentrations of membrane lipids are similar to those reported for the Central Pacific Ocean, supporting the recent estimation of M. B. Karner, E. F. DeLong, and D. M. Karl (Nature 409:507-510, 2001) that the world's oceans contain ca. 1028 cells of planktonic Crenarchaeota.
Soil nitrification is an important process for agricultural productivity and environmental pollution. Though one cultivated representative of ammonia-oxidizing Archaea from soil has been described, additional representatives warrant characterization. We describe an ammonia-oxidizing archaeon (strain MY1) in a highly enriched culture derived from agricultural soil. Fluorescence in situ hybridization microscopy showed that, after 2 years of enrichment, the culture was composed of >90% archaeal cells. Clone libraries of both 16S rRNA and archaeal amoA genes featured a single sequence each. No bacterial amoA genes could be detected by PCR. A [13C]bicarbonate assimilation assay showed stoichiometric incorporation of 13C into Archaea-specific glycerol dialkyl glycerol tetraethers. Strain MY1 falls phylogenetically within crenarchaeal group I.1a; sequence comparisons to “Candidatus Nitrosopumilus maritimus” revealed 96.9% 16S rRNA and 89.2% amoA gene similarities. Completed growth assays showed strain MY1 to be chemoautotrophic, mesophilic (optimum at 25°C), neutrophilic (optimum at pH 6.5 to 7.0), and nonhalophilic (optimum at 0.2 to 0.4% salinity). Kinetic respirometry assays showed that strain MY1's affinities for ammonia and oxygen were much higher than those of ammonia-oxidizing bacteria (AOB). The yield of the greenhouse gas N2O in the strain MY1 culture was lower but comparable to that of soil AOB. We propose that this new soil ammonia-oxidizing archaeon be designated “Candidatus Nitrosoarchaeum koreensis.”
A novel rod-shaped hyperthermophilic archaeum has been isolated from a boiling marine water hole at Maronti Beach, Ischia, Italy. It grew optimally at 100 degrees C and pH 7.0 by aerobic respiration as well as by dissimilatory nitrate reduction, forming dinitrogen as a final product. Organic and inorganic compounds served as substrates during aerobic and anaerobic respiration. Growth was inhibited by elemental sulfur. The cell wall was composed of a surface layer of hexameric protein complexes arranged on a p6 lattice. The core lipids consisted mainly of glycerol diphytanyl glycerol tetraethers with various degrees of cyclization. The G+C content was 52 mol%. The new isolate resembled members of the genera Thermoproteus and Pyrobaculum by its ability to form characteristic terminal spherical bodies ("golf clubs"). On the basis of its 16S rRNA sequence, the new isolate exhibited a close relationship to the genus Pyrobaculum. It is described as a new species, which we name Pyrobaculum aerophilum (type strain: IM2; DSM 7523).
Glycerol dialkyl glycerol tetraethers (GDGTs) found in hot springs reflect the abundance and community structure of Archaea in these extreme environments. The relationships between GDGTs, archaeal communities, and physical or geochemical variables are underexamined to date and when reported often result in conflicting interpretations. Here, we examined profiles of GDGTs from pure cultures of Crenarchaeota and from terrestrial geothermal springs representing a wide distribution of locations, including Yellowstone National Park (United States), the Great Basin of Nevada and California (United States), Kamchatka (Russia), Tengchong thermal field (China), and Thailand. These samples had temperatures of 36.5 to 87°C and pH values of 3.0 to 9.2. GDGT abundances also were determined for three soil samples adjacent to some of the hot springs. Principal component analysis identified four factors that accounted for most of the variance among nine individual GDGTs, temperature, and pH. Significant correlations were observed between pH and the GDGTs crenarchaeol and GDGT-4 (four cyclopentane rings, m/z 1,294); pH correlated positively with crenarchaeol and inversely with GDGT-4. Weaker correlations were observed between temperature and the four factors. Three of the four GDGTs used in the marine TEX86 paleotemperature index (GDGT-1 to -3, but not crenarchaeol isomer) were associated with a single factor. No correlation was observed for GDGT-0 (acyclic caldarchaeol): it is effectively its own variable. The biosynthetic mechanisms and exact archaeal community structures leading to these relationships remain unknown. However, the data in general show promise for the continued development of GDGT lipid-based physiochemical proxies for archaeal evolution and for paleo-ecology or paleoclimate studies.
Simulations of a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine lipid bilayer interacting with a solid surface of hydroxylated nanoporous amorphous silica have been carried out over a range of lipid-solid substrate distances. The porous solid surface allowed the water layer to dynamically adjust is thickness, maintaining equal pressures above and below the membrane bilayer. Qualitative estimates of the force between the surfaces leads to an estimated lipid-silicon distance in very good agreement with the results of neutron scattering experiments. Detailed analysis of the simulation at the separation suggested by experiment shows that for this type of solid support the water layer between surfaces is very narrow, consisting only of bound waters hydrating the lipid headgroups and hydrophilic silica surface. The reduced hydration, however, has only minor effects on the headgroup hydration, the orientation of water molecules at the interface, and the membrane dipole potential. While these structural properties were not sensitive to the presence of the solid substrate, the calculated diffusion coefficient for translation of the lipid molecules was altered significantly by the silica surface.
molecular dynamics; diffusion; lipid bilayer; solid support
Thermoplasma acidophilum HO-62 was grown at different pHs and temperatures, and its polar lipid compositions were determined. Although the number of cyclopentane rings in the caldarchaeol moiety increased when T. acidophilum was cultured at high temperature, the number decreased at low pHs. Glycolipids, phosphoglycolipids, and phospholipids were analyzed by high-performance liquid chromatography with an evaporative light-scattering detector. The amount of caldarchaeol with more than two sugar units on one side increased under low-pH and high-temperature conditions. The amounts of glycolipids increased and those of phosphoglycolipids decreased under these conditions. The proton permeability of the liposomes obtained from the phosphoglycolipids that contained two or more sugar units was lower than that of the liposomes obtained from the phosphoglycolipids that contained one sugar unit. From these results, we propose the hypothesis that T. acidophilum adapts to low pHs and high temperatures by extending sugar chains on their cell surfaces, as well as by varying the number of cyclopentane rings.
The kingdom Crenarchaeota is now known to include archaea which inhabit a wide variety of low-temperature environments. We report here lipid analyses of nonthermophilic crenarchaeotes, which revealed the presence of cyclic and acyclic dibiphytanylglycerol tetraether lipids. Nonthermophilic crenarchaeotes appear to be a major biological source of tetraether lipids in marine planktonic environments.